Title: Functional analysis of structure specific nucleases in genome stability and cancers Abstract The goal of this application is to provide essential support for my research on the molecular basis of genomic instability in cancer, which is conducted in the laboratory of Dr. Binghui Shen, Professor and Chair of Cancer Genetics and Epigenetics at the Beckman Research Institute of City of Hope. It is my intention to leverage the excellent resources available at City of Hope, and within an established laboratory, to maximize my ongoing contribution to science, rather than build an independent research program from the ground up. Dr. Shen's research program has been sponsored by two long-term continuously funded R01 grants, NCI R01 CA073764 (1997-2017) and NCI R01 CA085344 (1998-2018). Since I joined the Shen laboratory in 2001, I have co- authored 32 of the 46 peer reviewed articles that are associated with these two research programs, including manuscripts published in Nature Medicine, Molecular Cell, Nature Chemical Biology, and Nature Communications. As an Assistant Research Professor of Cancer Genetics and Epigenetics, I bring to the Shen laboratory expertise in protein biochemistry, cell culture assays of DNA repair, and mouse genetics, as well as mentoring, project management, and manuscript preparation. My major contributions to the field of DNA replication and repair, and their connection to cancer, include the biochemical characterization of mammalian FEN1 and DNA2 and demonstration of FEN1 and DNA2 mutations as cancer etiological factors. The goal of the NCI R01 CA073764 research program is to establish a comprehensive relationship among genetic alterations, functional deficiency, and pathological consequences, using FEN1 nuclease as a model protein and transgenic mice as a model system. During the previous funding cycles, we evaluated FEN1 mutations using cancer cells and transgenic knock-in mice that disrupt FEN1's nuclease activities, protein/protein interactions and post-translational modifications (PTMs), and contribute to tumorigenesis. In the current funding cycle, we are investigating the role of FEN1 PTMs in genome maintenance during DNA replication and their potential role in cancer. Meanwhile, the goal of the NCI R01 CA085344 research program is to define the roles of the DNA2 nuclease/helicase in genome integrity and tumor suppression. We have found that the role of DNA2 extends beyond the previously established role in Okazaki fragment maturation; it also stabilizes stalled DNA replication forks, repairs collapsed DNA replication forks, participates in telomere replication, regulates mtDNA repair, and regulates cancer development. We are currently testing the hypothesis that PTMs on mammalian DNA2 regulate its response to DNA replication stress from both exogenous and endogenous sources. As part of this project, I am establishing and testing new mouse models with DNA2 mutations that have been identified in human cancers. This funding support will allow me to continue my work on the established research programs in the Shen laboratory and will provide autonomy to pursue important novel lines of research that will form the basis of future collaborative funding proposals.